Significant evidence implicates increased consumption of omega-3 polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA), in the prevention of human diseases such as atherosclerosis. The mechanism by which DHA is protective is unknown although studies suggest that non-enzymatic oxidation products of DHA are anti-inflammatory. Nonetheless, these compounds have not been identified. In the previous cycle of this grant, we defined the free radical-initiated peroxidation of cholesteryl arachidonate in the context of atherosclerosis and have proposed a unified mechanism for its oxidation. The major peroxidation products consist of novel compounds containing mono- and serial cyclic peroxide and endoperoxide (isoprostane, IsoP) moieties. These compounds possess potent pro-inflammatory bioactivity and likely mediate various physiological and pathophysiological processes. Unlike arachidonate, the oxidation of DHA is predicted to be significantly more complex since it contains two additional carboncarbon unsaturated bonds. Studies proposed will examine the human pharmacology of DHA peroxidation in the context of atherosclerosis. We hypothesize the oxidation of DHA can be defined and results in the formation of compounds that contribute to the anti-inflammatory properties of this PUFA. The structural and mechanistic work will use docosahexaenoyl-glycerophosphatidylethanolamine (DHA-PE), because it is a major biologically relevant form and offers a unique opportunity to define the peroxidation of DHA in a physiologically relevant context. In addition, this will allow for the development of approaches to accurately characterize complex phospholipid oxidation products. A detailed study of the peroxidation of DHA, or for that matter any PUFA, esterifed in glycerophospholipids has not been undertaken. In Specific Aims 1 and 2, we will define mechanistically the free radical-initiated peroxidation of DHA-PE using novel chromatographic and mass spectrometric approaches. In Specific Aim 3, we will determine the effect of various factors, including novel antioxidants that we have developed, on the formation of different DHA-PE peroxidation products in vitro and in vivo. In Specific Aim 4, we will examine the inflammatory-mediating properties of DHA and selected peroxidation products. We will study the extent to which DHA decreases the formation of pro-inflammatory eicosanoids in animals and humans and also to what degree it reduces atherosclerosis in mouse models. Finally, we will examine the anti-inflammatory properties of one highly reactive DHA-derived cyclopentenone-containing IsoP-like compound formed in abundance in vivo, 4-A4t-neuroprostane. We contend that studies identifying novel oxidation products of DHA-PE and examining mechanisms by which these compounds are formed in vivo will yield insights into the role of this PUFA in human physiology and pathophysiology.